Abstract/Project Summary: Natural killer (NK) cells are important immune cells that protect the host from infection and cancer. They are homeostatically regulated by IL-15 trans-presented in the context of IL-15R?. We have established the anti-leukemic effect of allogeneic NK cell infusions, but host rejection of adoptively transferred NK cells and lack of NK cell specificity to tumor antigens limit their treatment success. During the current funding period we developed novel agents to target NK cells to tumors. These include bi-specific killer engagers (BiKEs), scFv anti-CD16 fusions with anti-CD33 or anti-PR1, and 15/IL-15R?-Fc complexes targeted to CD33. The goal of this proposal is to address relapse after non-myeloablative umbilical cord blood transplantation (UCBT), which is the major cause of treatment failure with relapse rates of 35%. Our data demonstrate that after UCBT NK cells are abundant but dysfunctional. However, individuals who activate cytomegalovirus (CMV) develop highly differentiated NKG2C+ NK cells. These cells, which we have named adaptive NK cells have an educated phenotype, are enriched for the expression of self-inhibitory KIRs and represent the human equivalent of the memory NK cells described in CMV-infected mice. Publications from our group established that after UCBT these adaptive NK cells have heightened effector functions that persist for at least one year. Our extensive preliminary data show that NKG2C is not the only marker for CMV-induced adaptive NK cells. We identified expansions of NK cells selectively lacking the proximal signaling molecules Fc?R1?, EAT-2 and SYK individually or in combination in UCBT recipients who reactivated CMV. Adaptive NK cells are epigenetically primed for enhanced cytokine production, survival, and increased expression of mTOR pathway adaptors. Further, they are functionally specialized for antibody-dependent cellular cytotoxicity (ADCC) through CD16. We will translate this research to develop minimally manipulative methods to activate NK cells and prolong their survival such as overnight ex vivo incubation with IL-15, IL-12 and IL-18. . We have also developed strategies to make NK cells antigen specific with the use of ?off-the-shelf? activation/targeting strategies. Our OVERARCHING HYPOTHESIS is that adaptive NK cells can be activated and targeted to induce antigen-specific killing to prevent or treat relapse after UCBT. In SA1, we will evaluate the clinical impact of IL-15 signaling on adaptive NK cells by analyzing samples from our biorepository with mature clinical data. We will also perform clinical trials in patients with hematologic malignancies undergoing non- myeloablative UCBT. The primary objective of these trials is to safely induce expansion of activated adaptive NK cells. In SA2, activation and antigen targeting of NK cells will be studied for prevention of relapse using an AML xenogeneic model to pick the best strategy to move into clinical testing in year 3. These targeted approaches include 1) IL-15/IL-15R?-Fc and anti-CD16x33 BiKE; 2) IL-15/IL-15R?-Fc-anti-CD33 complexes; or 3) IL-15/IL-15R?-Fc and anti-CD16xPR1 BiKE.